[Technical Field]
[0001] The present invention relates to
Lactobacillus paracasei-derived extracellular vesicles and a use thereof, and more particularly, to a composition
for preventing or treating an ocular disease, which comprises
Lactobacillus paracasei-derived extracellular vesicles as an active ingredient.
[0002] This application claims priority to and the benefit of
Korean Patent Application Nos. 10-2021-0072078 and
10-2021-0130843 filed in the Korean Intellectual Property Office on June 3, 2021 and October 1, 2021,
respectively, and all the contents disclosed in the specification and drawings of
the applications are incorporated in this application.
[Background Art]
[0003] Since the beginning of the 21st century, acute infectious diseases recognized as
epidemic diseases in the past have become less important, whereas chronic diseases
accompanied by immune dysfunction caused by disharmony between humans and microbiomes
have changed disease patterns as main diseases that determine the quality of life
and human lifespan. As 21
st century intractable chronic diseases characterized by abnormalities in immune and
metabolic functions caused by various stresses, cancer, cardiovascular diseases, chronic
lung diseases, metabolic diseases, and neuro-psychiatric diseases are major diseases
determining human lifespans and quality of life and becoming a big problem in public
health.
[0004] Immunity is a cellular defense mechanism against biological, chemical, physical and
mental stress, and occurs through innate immunity and adaptive immunity. Metabolism
is to make energy required for the body to produce various materials performing cell
functions, and provides proteins and lipids, which have been produced in the endoplasmic
reticulum (ER) by ATP produced in mitochondria, to a region in need thereof. Cells
face various stresses from the moment they are generated, and biological, chemical,
physical, and psychiatric stress induces ER stress in cells, and when the ER stress
is maintained, the cells die or are converted into cancer cells. Therefore, it has
been recently revealed that many intractable diseases, which are currently problematic,
are caused by abnormalities in immune and metabolic functions due to repetitive ER
stress.
[0005] Meanwhile, the retina of the eye is an organ belonging to the central nervous system,
and mature retinal cells do not divide under normal conditions like neuronal cells.
Accordingly, when the function of retinal cells decreases, it is easy to have abnormalities
in visual function, and aging rapidly progresses. The biggest cause of deteriorated
retinal cell function is oxidative stress, which is because tissues for constituting
the eye, including the retina, optic nerve, photoreceptor cells and lens, are constantly
exposed to oxidative stress such as light and UV in daily life. Due to such oxidative
stress, as the alteration of DNA, proteins and lipids constituting a cell occurs and
cell death is induced, ocular aging occurs, and seriously, age-related ocular diseases
such as geographic atrophy, diabetic retinopathy, cataracts, glaucoma and dry eye
occur.
[0006] In addition, when vision-related cells do not properly defend against environmental
stress such as blue light and UV, inflammation of eyes occurs, and chronic inflammatory
ocular disease occurs due to repeated stress. Recently, in order to treat or prevent
these chronic inflammatory ocular diseases, interest in an inhibitor of an inflammatory
cytokine, TNF-α, which is known as a major mediator of an inflammatory disease, is
increasing.
[0007] It is known that the number of microorganisms that coexist in the human body reaches
100 trillion, which is about 10-fold larger than that of human cells, and the number
of genes of microorganisms is 100-fold larger than that of humans. A microbiota or
microbiome refers to a microbial community including bacteria, archaea and eukarya
present in a given habitat.
[0008] Bacteria that coexist in our bodies and bacteria that exist in the surrounding environment
secrete nanometer-sized vesicles to exchange information such as genes, low molecular
compounds, and proteins with other cells. The mucosa forms a physical defense membrane
through which particles having a size of 200 nanometers (nm) or more cannot pass,
so that bacteria coexisting in the mucosa cannot pass through the mucosa, but bacteria-derived
extracellular vesicles have a size of approximately 20 to 200 nanometers, and thus
relatively freely pass through epithelial cells via the mucosa to be absorbed in our
bodies. As described above, although bacteria-derived extracellular vesicles are secreted
from bacteria, they differ from bacteria in terms of their constituents, absorption
rate in the body, and risk of side effects, and therefore, the use of bacteria-derived
extracellular vesicles is completely different from that of living cells or has a
significant effect.
[0009] Locally secreted bacterial-derived extracellular vesicles are absorbed through the
epithelial cells of the mucosa to induce a local inflammatory response, and vesicles
that have passed through the epithelial cells are systemically absorbed to be distributed
to respective organs, and regulate immune and inflammatory responses in the distributed
organs. For example, vesicles derived from pathogenic bacteria such as
Escherichia coli are pathogenic nanoparticles mimicking viruses, causing colitis or food poisoning
locally, and when absorbed into a blood vessel, promote systemic inflammatory responses
and blood coagulation through a vascular endothelial inflammatory response. On the
other hand, vesicles derived from beneficial bacteria may control diseases by regulating
abnormalities in immune and metabolic functions caused by pathogenic vesicles.
[0010] Lactic acid bacteria include a large number of bacteria converting carbohydrates
into lactic acid, and are known to ferment foods such as yogurt, lactic acid bacterial
drinks, and kimchi, to protect the body from other pathogenic microbes due to being
present in digestive organs (enteric bacteria) such as intestines or in the vagina,
and to help maintain homeostasis. Among the lactic acid bacteria,
Lactobacillus paracasei is aerobic lactic acid bacteria which do not form spores, and is known to serve as
symbiotic bacteria in the human gastrointestinal tract and a woman's vagina to protect
the human body from pathogenic bacteria.
[0011] However, there is no reported case of application of
Lactobacillus paracasei-derived extracellular vesicles to treat age- or inflammation-related ocular diseases.
[Disclosure]
[Technical Problem]
[0012] To solve the problems in the conventional art described above, the present invention
is directed to providing a pharmaceutical composition for preventing or treating an
ocular disease, comprising
Lactobacillus paracasei-derived extracellular vesicles as an active ingredient.
[0013] In addition, another object of the present invention is to provide a food composition
for preventing or alleviating an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0014] In addition, still another object of the present invention is to provide an quasi-drug
composition for preventing or treating an ocular disease, comprising vesicles derived
from
Lactobacillus paracasei as an active ingredient.
[0015] In addition, still another object of the present invention is to provide an inhalable
composition for preventing or treating an ocular disease, comprising vesicles derived
from
Lactobacillus paracasei as an active ingredient.
[0016] In addition, still another object of the present invention is to provide a composition
for drug delivery for treating a ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0017] However, a technical problem to be achieved by the present invention is not limited
to the aforementioned problems, and the other problems that are not mentioned may
be clearly understood by a person skilled in the art from the following description.
[Technical Solution]
[0018] The present invention provides a pharmaceutical composition for preventing or treating
an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0019] In addition, the present invention provides a food composition for preventing or
alleviating an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0020] In addition, the present invention provides a quasi-drug composition for preventing
or alleviating an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0021] In addition, the present invention provides an inhalable composition for preventing
or alleviating an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0022] In addition, the present invention provides a composition for drug delivery for treating
a ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient.
[0023] In one embodiment of the present invention, the ocular disease may be an age-related
ocular disease, but the present invention is not limited thereto.
[0024] In another embodiment of the present invention, the ocular disease may be inflammatory
ocular disease, but the present invention is not limited thereto.
[0025] In still another embodiment of the present invention, the ocular disease may be one
or more diseases selected from the group consisting of Leber congenital amaurosis
(LCA), Stargardt's disease, Usher syndrome, choroidal insufficiency, Rod-Cone or Cone-Rod
dystrophy, ciliopathy, a mitochondrial disorder, progressive retinal atrophy, a degenerative
retinal disease, age-related macular degeneration (AMD), wet AMD, dry AMD, map atrophy,
familial or acquired maculopathy, a retinal photoreceptor disease, a retinal pigment
epithelium-related disease, diabetic retinopathy, cystic macular edema, retinal detachment,
traumatic retinal damage, iatrogenic retinal damage, a macular hole, macular telangiectasia,
a ganglion cell disease, an optic nerve cell disease, glaucoma, a cataract, optic
neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vessel occlusion,
familial macroaneurism, a retinal vascular disease, an ophthalmic vascular disease,
retinal nerve cell degeneration caused by glaucoma and ischemic optic neuropathy,
but the present invention is not limited thereto.
[0026] In still another embodiment of the present invention, the inflammatory ocular disease
may be one or more diseases selected from the group consisting of retinitis pigmentosa
(RP), conjunctivitis, scleritis, keratitis, iritis, uveitis, chorioretinitis, choroiditis,
and retinitis, but the present invention is not limited thereto.
[0027] In still another embodiment of the present invention, the inflammatory ocular disease
may be mediated by IL-6, but the present invention is not limited thereto.
[0028] In yet another embodiment of the present invention, the ocular disease may be an
ocular disease caused by immune dysfunction or metabolic dysfunction, but the present
invention is not limited thereto.
[0029] In yet another embodiment of the present invention, the ocular disease may be an
ocular disease caused by oxidative stress, but the present invention is not limited
thereto.
[0030] In yet another embodiment of the present invention, the vesicles may have an average
diameter of 10 to 300 nm, but the present invention is not limited thereto.
[0031] In yet another embodiment of the present invention, the vesicles may be naturally
or artificially secreted from
Lactobacillus paracasei, but the present invention is not limited thereto.
[0032] In yet another embodiment of the present invention, the vesicles may be isolated
from a
Lactobacillus paracasei culture solution or a food prepared by adding
Lactobacillus paracasei, but the present invention is not limited thereto.
[0033] Further, the present invention provides a method for preventing or treating an ocular
disease, the method comprising administering a composition comprising vesicles derived
from
Lactobacillus paracasei to a subject.
[0034] In addition, the present invention provides a use of a composition comprising vesicles
derived from
Lactobacillus paracasei for preventing or treating an ocular disease.
[0035] Further, the present invention provides a use of vesicles derived from
Lactobacillus paracasei for preparing a drug for preventing or treating an ocular disease.
[0036] In addition, the present invention provides a use of vesicles derived from
Lactobacillus paracasei for preparing a food for preventing or alleviating an ocular disease.
[0037] Further, the present invention provides a use of vesicles derived from
Lactobacillus paracasei for preparing a quasi-drug for preventing or alleviating an ocular disease.
[0038] In addition, the present invention provides a method for delivery of drug for treating
an ocular disease, the method comprising administering a composition comprising vesicles
derived from
Lactobacillus paracasei to a subject.
[0039] Further, the present invention provides a use of a composition comprising vesicles
derived from
Lactobacillus paracasei for delivery of drug for treating an ocular disease.
[0040] In addition, the present invention provides use of vesicles derived from
Lactobacillus paracasei for preparing a drug delivery agent for treating an ocular disease.
[Advantageous Effects]
[0041] It was confirmed that, after being absorbed into the body,
Lactobacillus paracasei-derived extracellular vesicles according to the present invention are distributed to the
central nervous system through the blood brain barrier (BBB), and after being absorbed
into cells, effectively inhibit the secretion of IL-6, which is the main mediator
of intractable inflammatory diseases, activate AMPK signaling to increase metabolic
function, and efficiently inhibit the occurrence of an ocular disease caused by cell
senescence and inflammation. Further, as it was confirmed that, when the vesicles
were administered into a rabbit model with an ocular disease caused by oxidative stress,
the occurrence of retinal degeneration caused by oxidative stress was significantly
inhibited, the
Lactobacillus paracasei-derived extracellular vesicles according to the present invention can be expected to be widely
used as a drug for alleviating, preventing or treating an ocular disease.
[Description of Drawings]
[0042]
FIG. 1 is a result of analyzing the distribution pattern of Lactobacillus paracasei-derived extracellular vesicles after oral administration according to one embodiment of the
present invention.
FIG. 2 is a result of analyzing the distribution pattern of Lactobacillus paracasei -derived extracellular vesicles in the central nervous system after oral administration
according to one embodiment of the present invention.
FIG. 3 is a graph evaluating the inhibitory effect of Lactobacillus paracasei-derived extracellular vesicles on the influence of inflammatory cytokine secretion by LPS
according to one embodiment of the present invention and the therapeutic effect of
the vesicles in heat treatment, acid treatment or bile treatment of the Lactobacillus paracasei-derived extracellular vesicles (MDH-001: Lactobacillus paracasei EVs).
FIG. 4 shows a result of assessing the therapeutic efficacy of Lactobacillus paracasei-derived extracellular vesicles on a metabolic function disorder according to one embodiment
of the present invention, and evaluating the effect on AMPK signaling which is the
key signaling pathway of metabolic function by treatment of cells with metformin as
a control drug and Lactobacillus paracasei-derived extracellular vesicles (MDH-001: Lactobacillus paracasei EVs).
FIG. 5 shows an animal model test and evaluation methods for confirming the efficacy
on an ocular disease by oral administration of Lactobacillus paracasei-derived extracellular vesicles according to one embodiment of the present invention.
FIG. 6 is a diagram showing a result obtained by measuring a retinal degenerated area
of treatment groups compared to a control by oral administration of Lactobacillus paracasei-derived extracellular vesicles into a rabbit model with an ocular disease in order to determine
the efficacy on the ocular disease. G1: untreated negative control, G2: disease-induced
positive control, G3: low dose vesicle-treated group, G4: high dose vesicle-treated
group.
FIG. 7 is a set of photographs of the fundus taken with a fundus camera (TRC-50IX,
TOPCON, Japan) after oral administration of Lactobacillus paracasei-derived extracellular vesicles into a rabbit model with an ocular disease caused by oxidative
stress in order to determine the efficacy on an age-related ocular disease. G1: untreated
negative control, G2: disease-induced positive control, G3: low dose vesicle-treated
group, G4: high dose vesicle-treated group.
FIG. 8 is a diagram illustrating an action mechanism of Lactobacillus paracasei-derived extracellular vesicles for an ocular disease according to one embodiment of the present
invention.
[Best modes of the Invention]
[0043] The present invention relates to
Lactobacillus paracasei-derived extracellular vesicles and a use thereof.
[0044] The present inventors isolated the vesicles derived from Lactobacillus paracasei.
[0045] The inventors confirmed that
Lactobacillus paracasei-derived extracellular vesicles are well delivered to several organs after oral administration
of the vesicles, and are migrated and distributed in the central nervous system including
the retina through the blood-brain barrier (BBB) (see Example 2).
[0046] In addition, the inventors confirmed that
Lactobacillus paracasei-derived extracellular vesicles inhibited IL-6 secretion in response to inflammation caused
by LPS, and such an inhibitory effect is maintained by heat treatment, acid treatment,
and bile treatment on the vesicles (see Example 3).
[0047] In addition, the inventors confirmed that, as autophagy is induced by activating
AMPK signaling, which is a key signaling factor of regulation of a metabolic disorder,
by
Lactobacillus paracasei-derived extracellular vesicles, an ocular disease caused by a metabolic disorder
can be treated by increasing the homeostasis of metabolic function (see Example 4).
[0048] In addition, the inventors confirmed that, as a result of oral administration of
Lactobacillus paracasei-derived extracellular vesicles to a rabbit model with macular degeneration caused
by oxidative stress, macular degeneration caused by the degeneration of the retinal
pigment epithelial cell layer is significantly reduced (see Example 5).
[0049] Thus, the present invention provides a composition for preventing, alleviating, or
treating an ocular disease, comprising vesicles derived from
Lactobacillus paracasei as an active ingredient. The composition includes a pharmaceutical composition, a
food composition, and a quasi-drug composition.
[0050] Hereinafter, a use of
Lactobacillus paracasei-derived extracellular vesicles of the present invention for preventing, alleviating or treating
an ocular disease will be described in detail.
[0051] As used herein, the term vesicle or extracellular vesicle refers to a structure formed
of a nano-sized membrane secreted from various bacteria, and includes, for example,
an extracellular vesicle derived from gram-negative bacteria such as
E. coli, which has, an endotoxin (lipopolysaccharide), a toxic protein, and both bacterial
DNA and RNA, or an extracellular vesicle derived from gram-positive bacteria such
as micrococcus bacteria, which have outer membrane vesicles (OMVs), a protein and
a nucleic acid as well as components of a bacterial cell wall, such as peptidoglycan
and lipoteichoic acid.
[0052] In the specification, the vesicle encompasses all structures which are naturally
secreted from
Lactobacillus paracasei, or formed of an artificially produced membrane.
[0053] The vesicles may be isolated by heat treatment or autoclaving during
Lactobacillus paracasei culture, or using one or more methods selected from the group consisting of centrifugation,
ultracentrifugation, autoclaving, extrusion, sonication, cell lysis, homogenization,
freezing-thawing, electroporation, mechanical degradation, chemical treatment, filtration
with a filter, gel filtration chromatography, pre-flow electrophoresis, and capillary
electrophoresis of the cell culture. In addition, for isolation, washing for removing
impurities, and concentration of the obtained vesicles may be further performed.
[0054] The method for isolating vesicles from the culture solution or fermented food of
the Lactobacillus paracasei of the present invention is not particularly limited as
long as the vesicles are included. For example, vesicles may be isolated using a method
such as centrifugation, ultrahigh speed centrifugation, filtration by a filter, gel
filtration chromatography, free-flow electrophoresis, or capillary electrophoresis,
and a combination thereof, and further, a process such as washing to remove impurities
and concentration of obtained vesicles may be further included.
[0055] The term "ocular disease" used herein refers to an eye-related disease.
[0056] The term "age-related ocular disease" used herein is a concept that includes not
only an ocular disease caused by degradation of biological functions with aging, but
also an ocular disease exhibiting similar symptoms to those of a disease mainly occurring
in the elderly due to degradation of biological functions faster than an actual age.
In the present invention, the age-related ocular disease may include an ocular disease
mediated by AMPK and an ocular disease caused by oxidative stress. The age-related
ocular disease may comprise, for example, one or more selected from the group consisting
of Leber congenital amaurosis (LCA), Stargardt's disease, Usher syndrome, choroidal
insufficiency, Rod-Cone or Cone-Rod dystrophy, ciliopathy, a mitochondrial disorder,
progressive retinal atrophy, a degenerative retinal disease, age-related macular degeneration
(AMD), wet AMD, dry AMD, map atrophy, familial or acquired maculopathy, a retinal
photoreceptor disease, a retinal pigment epithelium-related disease, diabetic retinopathy,
cystic macular edema, retinal detachment, traumatic retinal damage, iatrogenic retinal
damage, a macular hole, macular telangiectasia, a ganglion cell disease, an optic
nerve cell disease, glaucoma, a cataract, optic neuropathy, ischemic retinal disease,
retinopathy of prematurity, retinal vessel occlusion, familial macroaneurism, a retinal
vascular disease, an ophthalmic vascular disease, retinal nerve cell degeneration
caused by glaucoma and ischemic optic neuropathy, but the present invention is not
limited thereto.
[0057] The term "inflammatory ocular disease" used herein is a concept including all ocular
diseases caused by inflammation in the eye due to an inflammatory causative factor,
and in the present invention, the inflammatory ocular disease may include an ocular
disease mediated by IL-6 or an ocular disease caused by oxidative stress. The inflammatory
ocular disease may include, for example, retinitis pigmentosa (RP), conjunctivitis,
scleritis, keratitis, iritis, uveitis, chorioretinitis, choroiditis, and retinitis,
but the present invention is not limited thereto.
[0058] The "retinal geographic atrophy" used herein is a disease in which the retina and
choriocapillaris atrophy due to calcification of drusen, which are waste accumulating
in the retinal pigment epithelium, due to age-related macular degeneration and poor
blood supply, and the atrophied area enlarges in a map shape and spreads to the central
region, resulting in loss of vision.
[0059] The "age-related macular degeneration (AMD)" used herein is a disease accompanied
by various changes due to aging in the macula of the retina, which plays a very important
role in vision. In the present invention, the age-related macular degeneration may
comprise dry (non-exudative) and wet (exudative) types. The "dry AMD" refers to the
case in which there is a lesion such as drusen or retinal pigment epithelium atrophy
in the retina, and accounts for almost 90% of AMD The photoreceptor cells in the macula
gradually atrophy, vision gradually decreases, and it can develop into a wet form.
In the "wet AMD," new choroidal blood vessels grow under the retina, and severe visual
damage is likely to occur due to bleeding or exudation from the new blood vessel itself
or blood vessel, and blindness may occur due to disc atrophy and severe hemorrhaging
within months to years after onset.
[0060] The "diabetic retinopathy" used herein is one of the microvascular complications
appearing in diabetes, and is caused by functional and morphological changes in capillaries
such as increased vascular permeability in the retina due to high blood pressure and
the accompanying several biochemical changes, ischemia and neovascularization. In
the present invention, the diabetic retinopathy may comprise non-proliferative or
proliferative retinopathy, but the present invention is not limited thereto. The "non-proliferative
retinopathy" refers to a condition in which small blood vessels in the retina are
weakened and the supply of nutrients is interrupted due to serum leakage or blockage
of blood vessels. The "proliferative retinopathy" is known to cause blindness within
5 years due to bleeding from a new blood vessel without suitable treatment for neovascularization
in a place with poor blood circulation.
[0061] In the present invention, the vesicles may have the form of a spherical shape, and
have an average diameter 10-300 nm, 10-200 nm, 10-190 nm, 10-180 nm, 10-170 nm, 10-160
nm, 10-150 nm, 10-140 nm, 10-130 nm, 10-120 nm, 10-110 nm, 10-100 nm, 10-90 nm, 10-80
nm, 10-70 nm, 10-60 nm, 10-50 nm, 20-200 nm, 20-180 nm, 20-160 nm, 20 to 140 nm, 20
to 120 nm, 20 to 100 nm, or 20 to 80 nm, preferably 20-200 nm, but is not limited
thereto.
[0062] The term "comprising as an active ingredient" used herein refers to a sufficient
amount for achieving the efficacy or activity of
Lactobacillus paracasei-derived extracellular vesicles.
[0063] The amount of the vesicles in the composition of the present invention may be appropriately
adjusted depending on the symptoms of a disease, the degree of progression of symptoms,
the condition of a patient, and the like, and may range from, for example, 0.0001
wt% to 99.9 wt% or 0.001 wt% to 50 wt% with respect to a total weight of the composition,
but the present invention is not limited thereto. The amount ratio is a value based
on the amount of dried product from which a solvent is removed.
[0064] The pharmaceutical composition according to the present invention may further include
a suitable carrier, excipient, and diluent which are commonly used in the preparation
of pharmaceutical compositions. The excipient may be, for example, one or more selected
from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an
adsorbent, a humectant, a film-coating material, and a controlled release additive.
[0065] The pharmaceutical composition according to the present invention may be used by
being formulated, according to commonly used methods, into a form such as powders,
granules, sustained-release-type granules, enteric granules, liquids, eye drops, elixirs,
emulsions, suspensions, spirits, troches, aromatic water, lemonades, tablets, sustained-release-type
tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release-type
capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts,
injections, capsules, perfusates, or a preparation for external use, such as plasters,
lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols.
The preparation for external use may have a formulation such as creams, gels, patches,
sprays, ointments, plasters, lotions, liniments, pastes, or cataplasmas.
[0066] As the carrier, the excipient, and the diluent that may be included in the pharmaceutical
composition according to the present invention, lactose, dextrose, sucrose, oligosaccharides,
sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate,
gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline
cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate,
talc, magnesium stearate, and mineral oil may be used.
[0067] For formulation, commonly used diluents or excipients such as fillers, thickeners,
binders, wetting agents, disintegrants, and surfactants are used.
[0068] As additives of tablets, powders, granules, capsules, pills, and troches according
to the present invention, excipients such as corn starch, potato starch, wheat starch,
lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate,
synthetic aluminum silicate, dibasic calcium phosphate, calcium sulfate, sodium chloride,
sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin,
sodium alginate, methyl cellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal
silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC
2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and
Primojel® and binders such as gelatin, Arabic gum, ethanol, agar powder, cellulose acetate
phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified
water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium
methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose,
hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone
may be used, and disintegrants such as hydroxypropyl methylcellulose, corn starch,
agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose,
sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate,
silicic anhydride, 1-hydroxypropylcellulose, dextran, ion-exchange resin, polyvinyl
acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum,
sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, Arabic
gum, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium
aluminum silicate, a di-sorbitol solution, and light anhydrous silicic acid; and lubricants
such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable
oil, talc, lycopodium powder, kaolin, Vaseline, sodium stearate, cacao butter, sodium
salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid
paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate,
sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic
anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene
glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine,
and light anhydrous silicic acid may be used.
[0069] As additives of liquids according to the present invention, water, dilute hydrochloric
acid, dilute sulfuric acid, sodium citrate, monostearic acid sucrose, polyoxyethylene
sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin
ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate,
potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose,
and sodium carboxymethylcellulose may be used.
[0070] In syrups according to the present invention, a white sugar solution, other sugars
or sweeteners, and the like may be used, and as necessary, a fragrance, a colorant,
a preservative, a stabilizer, a suspending agent, an emulsifier, a viscous agent,
or the like may be used.
[0071] In emulsions according to the present invention, purified water may be used, and
as necessary, an emulsifier, a preservative, a stabilizer, a fragrance, or the like
may be used.
[0072] In suspensions according to the present invention, suspending agents such as acacia,
tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose,
microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC)
1828, HPMC 2906, HPMC 2910, and the like may be used, and as necessary, a surfactant,
a preservative, a stabilizer, a colorant, and a fragrance may be used.
[0073] Injections according to the present invention may include: solvents such as distilled
water for injection, a 0.9% sodium chloride solution, Ringer's solution, a dextrose
solution, a dextrose+sodium chloride solution, PEG, lactated Ringer's solution, ethanol,
propylene glycol, non-volatile oil-sesame oil, cottonseed oil, peanut oil, soybean
oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; cosolvents
such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide,
butazolidine, propylene glycol, the Tween series, amide nicotinate, hexamine, and
dimethylacetamide; buffers such as weak acids and salts thereof (acetic acid and sodium
acetate), weak bases and salts thereof (ammonia and ammonium acetate), organic compounds,
proteins, albumin, peptone, and gums; isotonic agents such as sodium chloride; stabilizers
such as sodium bisulfite (NaHSO
3) carbon dioxide gas, sodium metabisulfite (Na
2S
2O
5), sodium sulfite (Na
2SO
3), nitrogen gas (N
2), and ethylenediamine tetraacetic acid; sulfating agents such as 0.1% sodium bisulfide,
sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and
acetone sodium bisulfite; a pain relief agent such as benzyl alcohol, chlorobutanol,
procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such
as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.
[0074] In suppositories according to the present invention, bases such as cacao butter,
lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose,
a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm
oil, cacao butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween
or span, imhausen, monolan(propylene glycol monostearate), glycerin, Adeps solidus,
buytyrum Tego-G, cebes Pharma 16, hexalide base 95, cotomar, Hydrokote SP, S-70-XXA,
S-70-XX75(S-70-XX95), Hydrokote 25, Hydrokote 711, idropostal, massa estrarium (A,
AS, B, C, D, E, I, T), masa-MF, masupol, masupol-15, neosuppostal-N, paramount-B,
supposiro OSI, OSIX, A, B, C, D, H, L, suppository base IV types AB, B, A, BC, BBG,
E, BGF, C, D, 299, suppostal N, Es, Wecoby W, R, S, M, Fs, and tegester triglyceride
matter (TG-95, MA, 57) may be used.
[0075] Solid preparations for oral administration include tablets, pills, powders, granules,
capsules, and the like, and such solid preparations are formulated by mixing the composition
with at least one excipient, e.g., starch, calcium carbonate, sucrose, lactose, gelatin,
and the like. In addition to simple excipients, lubricants such as magnesium stearate
and talc are also used.
[0076] Examples of liquid preparations for oral administration include suspensions, liquids
for internal use, emulsions, syrups, and the like, and these liquid preparations may
include, in addition to simple commonly used diluents, such as water and liquid paraffin,
various types of excipients, for example, a wetting agent, a sweetener, a fragrance,
a preservative, and the like. Preparations for parenteral administration include an
aqueous sterile solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried
preparation, and a suppository. Non-limiting examples of the non-aqueous solvent and
the suspension include propylene glycol, polyethylene glycol, a vegetable oil such
as olive oil, and an injectable ester such as ethyl oleate.
[0077] The pharmaceutical composition according to the present invention is administered
in a pharmaceutically effective amount. In the present invention, "the pharmaceutically
effective amount" refers to an amount sufficient to treat diseases at a reasonable
benefit/risk ratio applicable to medical treatment, and an effective dosage level
may be determined according to factors including types of diseases of patients, the
severity of disease, the activity of drugs, sensitivity to drugs, administration time,
administration route, excretion rate, treatment period, and simultaneously used drugs,
and factors well known in other medical fields.
[0078] The composition according to the present invention may be administered as an individual
therapeutic agent or in combination with other therapeutic agents, may be administered
sequentially or simultaneously with therapeutic agents in the related art, and may
be administered in a single dose or multiple doses. It is important to administer
the composition in a minimum amount that can obtain the maximum effect without any
side effects, in consideration of all the aforementioned factors, and this may be
easily determined by those of ordinary skill in the art.
[0079] The pharmaceutical composition of the present invention may be administered to an
a subject via various routes. All administration methods can be predicted, and the
pharmaceutical composition may be administered via, for example, oral administration,
subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular
injection, intrathecal (space around the spinal cord) injection, sublingual administration,
administration via the buccal mucosa, intrarectal insertion, intravaginal insertion,
ocular administration, intra-aural administration, intranasal administration, inhalation,
spraying via the mouth or nose, transdermal administration, percutaneous administration,
or the like.
[0080] In the present invention, the ocular administration may be one selected from the
group consisting of intraconjunctival administration, intravitreal administration,
subretinal administration, suprachoroidal administration, subconjunctival administration
and sub-tenon administration, but the present invention is not limited thereto.
[0081] The pharmaceutical composition of the present invention is determined depending on
the type of a drug, which is an active ingredient, along with various related factors
such as a disease to be treated, administration route, the age, gender, and body weight
of a patient, and the severity of diseases. Specifically, the effective amount of
the composition according to the present invention may vary depending on the patient's
age, sex, and body weight, and generally, 0.001 to 150 mg of the composition and preferably,
0.01 to 100 mg of the composition, per 1 kg of the body weight, may be administered
daily or every other day or may be administered once to three times a day. However,
since the effective amount may be increased or decreased depending on the administration
route, the severity of obesity, gender, body weight, age, and the like, the dosage
is not intended to limit the scope of the present invention in any way.
[0082] As used herein, the "subject" refers to a subject in need of treatment of a disease,
and more specifically, refers to a mammal such as a human or a non-human primate,
a mouse, a rat, a dog, a cat, a horse, and a cow, but the present invention is not
limited thereto.
[0083] As used herein, the "administration" refers to providing a subject with a predetermined
composition of the present invention by using an arbitrary appropriate method.
[0084] The term "prevention" as used herein means all actions that inhibit or delay the
onset of a target disease. The term "treatment" as used herein means all actions that
alleviate or beneficially change a target disease and abnormal metabolic symptoms
caused thereby via administration of the pharmaceutical composition according to the
present invention. The term "improvement" as used herein means all actions that reduce
the degree of parameters related to a target disease, e.g., symptoms via administration
of the composition according to the present invention.
[0085] In addition, the present invention provides a food composition comprising vesicles
derived from
Lactobacillus paracasei as an active ingredient.
[0086] The food composition may be a health functional food composition, but is not limited
thereto.
[0087] The vesicles according to the present invention may be used by adding an active ingredient
as is to food or may be used together with other foods or food ingredients, but may
be appropriately used according to a typical method. The mixed amount of the active
ingredient may be suitably determined depending on the purpose of use thereof (for
prevention or alleviation). In general, when a food or beverage is prepared, the composition
of the present invention is added in an amount of 15 wt% or less, preferably 10 wt%
or less based on the raw materials. However, for long-term intake for the purpose
of health and hygiene or for the purpose of health control, the amount may be less
than the above-mentioned range, and the vesicles have no problem in terms of stability,
so the active ingredient may be used in an amount more than the above-mentioned range.
[0088] The type of food is not particularly limited. Examples of food to which the material
may be added include meats, sausage, bread, chocolate, candies, snacks, confectioneries,
pizza, instant noodles, other noodles, gums, dairy products including ice creams,
various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and
the like, and include all health functional foods in a typical sense.
[0089] The health beverage composition according to the present invention may contain various
flavors or natural carbohydrates, and the like as additional ingredients as in a typical
beverage. The above-described natural carbohydrates may be monosaccharides such as
glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such
as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
As a sweetener, it is possible to use a natural sweetener such as thaumatin and stevia
extract, a synthetic sweetener such as saccharin and aspartame, and the like. The
proportion of the natural carbohydrates is generally about 0.01 to 0.20 g, or about
0.04 to 0.10 g per 100 ml of the composition of the present invention.
[0090] In addition to the aforementioned ingredients, the composition of the present invention
may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic
acids and salts thereof, alginic acid and salts thereof, organic acids, protective
colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols,
carbonating agents used in carbonated drinks, and the like. In addition, the composition
of the present invention may contain flesh for preparing natural fruit juice, fruit
juice drinks, and vegetable drinks. These ingredients may be used either alone or
in combinations thereof. The proportion of these additives is not significantly important,
but is generally selected within a range of 0.01 to 0.20 part by weight per 100 parts
by weight of the composition of the present invention.
[0091] Further, the present invention may be provided in the form of an inhalable composition
comprising
Lactobacillus paracasei-derived vesicles as an active ingredient.
[0092] In the case of a preparation for inhalation, the compound may be formulated according
to a method known in the art, and may be conveniently delivered in the form of an
aerosol spray from a pressurized pack or a nebulizer by using a suitable propellant,
for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide, or other suitable gases. In the case of the pressurized aerosol, a
dosage unit may be determined by providing a valve for transferring a metered amount.
For example, a gelatin capsule and a cartridge for use in an inhaler or insufflator
may be formulated so as to contain a powder mixture of a compound and a suitable powder
base such as lactose or starch.
[0093] In addition, the present invention may provide a quasi-drug composition comprising
Lactobacillus paracasei-derived extracellular vesicles as an active ingredient.
[0094] The term "quasi-drug" used herein means products exhibiting a milder action than
pharmaceuticals among products used for diagnosing, curing, improving, alleviating,
treating or preventing a human or animal disease. For example, according to the Pharmaceutical
Affairs Act, the quasi-drugs exclude products used as pharmaceuticals, and include
textile • rubber products used for treating or preventing human • animal diseases,
products which act weakly or do not act directly on the human body, and are not instruments
or machines or similar thereto, and sterilizers and insecticides for preventing infectious
diseases.
[0095] In the present invention, the quasi-drug composition may be formulated as an ophthalmic
composition, for example, one or more selected from the group consisting of ophthalmic
liquids, eye drops, eye ointments, injection solutions, and eyewashes, but the present
invention is not limited thereto.
[0096] In addition, the present invention provides a composition for delivering a drug for
treating an ocular disease, which comprises
Lactobacillus paracasei-derived extracellular vesicles as an active ingredient.
[0097] The term "drug delivery" used herein means any means or act of loading and delivering
a drug to the composition according to the present invention in order to deliver a
drug to a specific organ, tissue, cell or cell organelle.
[Modes of the Invention]
[0098] Hereinafter, preferred Examples for helping the understanding of the present invention
will be suggested. However, the following Examples are provided only to more easily
understand the present invention, and the contents of the present invention are not
limited by the following Examples.
[Examples]
Example 1: Isolation of vesicles derived from Lactobacillus paracasei
[0099] To
isolate Lactobacillus paracasei-derived extracellular vesicles (EVs), various
Lactobacillus paracasei were inoculated into a self-made medium, cultured at 37 °C and 200 rpm until the
absorbance (OD
600nm) became 1.0 to 1.5, followed by re-inoculation in the medium and culturing. Then,
a supernatant from which bacterial cells had been removed was obtained by recovering
the culture solution including bacterial cells and performing centrifugation at 4°C
and 10,000 g for 20 minutes. The obtained supernatant was again filtered using a 0.22
µm filter, and the filtered supernatant was concentrated to a volume of 50 mL or less
using a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore) and a MasterFlex
pump system (Cole-Parmer). A vesicle derived from
Lactobacillus paracasei (MDH-001) was isolated by filtering the concentrated supernatant again using a 0.22
µm filter. The amount of protein included in the supernatant was measured using the
Pierce BCA Protein Assay kit (Thermo Fisher Scientific). The following experiments
for the isolated vesicles were conducted.
Example 2. Evaluation of pharmacokinetic characteristics of vesicles derived from
Lactobacillus paracasei bacteria
[0100] In order to investigate the pharmacokinetic characteristics of Lactobacillus paracasei-derived
vesicles during oral administration, the fluorescence expressed in the body and each
organ from immediately before administration to 72 hours after administration was
measured by orally administering vesicles stained with a fluorescent staining reagent
Cy7-NHS to mice.
[0101] As shown in FIG. 1, the
Lactobacillus paracasei-derived extracellular vesicles stained with fluorescence were orally administered, and after
1 hour, a fluorescent signal was observed. It can be confirmed that, at 3 hours, weak
signal levels were recorded in the stomach, small intestine, large intestine and lungs,
and at 6 hours, signals are shown in the brain as well as the stomach, small intestine,
large intestine and lungs. Such fluorescence signals showed the strongest signals
between 6 and 24 hours after oral administration, and showed a tendency to gradually
decrease, and almost disappeared after 48 hours.
[0102] In addition, as shown in FIG. 2, it was observed that, when the
Lactobacillus paracasei-derived extracellular vesicles are orally administered, and then a distribution pattern
in the central nervous system was evaluated, they were distributed in the central
nervous system 3 hours after oral administration, showed a peak at 6 hours, and the
signals disappeared after 48 hours.
[0103] From the result, it can be seen that, when orally administered, the
Lactobacillus paracasei-derived extracellular vesicles were absorbed into the body through a mucous membrane,
and distributed in various organs, and particularly, they migrated to the central
nervous system including the retina through the BBB and distributed.
Example 3: Evaluation of anti-inflammatory effect of Lactobacillus paracasei-derived extracellular vesicles on inflammatory reaction caused by LPS
[0104] To confirm the anti-inflammatory effect of the
Lactobacillus paracasei-derived extracellular vesicles, mouse macrophages (RAW 264.7 cells) were pre-treated with
the
Lactobacillus paracasei-derived extracellular vesicles at 10 µg/mL or 100 µg/mL, and treated with 100 ng/mL of LPS,
which is an inflammation inducer, followed by measuring a secretion amount of IL-6,
which is an inflammation-related marker. In addition, to evaluate whether the
Lactobacillus paracasei-derived extracellular vesicles can be orally administered, heat treatment, acid treatment
and bile treatment were performed on the vesicles, and then the secretion amount of
IL-6 was measured.
[0105] As a result, as shown in FIG. 3, compared to a positive control, which is a group
treated with LPS 100 ng/ml of LPS, it was confirmed that the
Lactobacillus paracasei-derived extracellular vesicles significantly reduce the secretion amount of IL-6. In addition,
it was confirmed that, in the heat treatment, acid treatment and bile treatment of
the
Lactobacillus paracasei-derived extracellular vesicles, the effect of inhibiting IL-6 secretion from macrophages
was also maintained.
[0106] The above results show that the
Lactobacillus paracasei-derived extracellular vesicles are stable against heat or an acid, and when the vesicles
are orally administered, an anti-inflammatory effect can be maintained.
Example 4: Evaluation of effect of Lactobacillus paracasei-derived extracellular vesicles on activation of AMPK signaling, which is key signaling that
regulates metabolic disorder
[0107] AMP-activated protein kinase (AMPK) signaling inhibits a metabolic disorder occurring
in energy depletion through a mechanism such as autophagy. In the present invention,
cells were treated with
Lactobacillus paracasei-derived extracellular vesicles (MDH-001-CM) to evaluate an effect on AMPK activation. DMEM
serum-free media into which 2 × 10
6 of the cells were seeded were put into a 60-mm cell culture petri dish and cultured
for 2 hours. Afterward, the
Lactobacillus paracasei-derived extracellular vesicles were treated at 0, 0.1, 1 or 10 µg/mL for 1 hour, and were
treated with insulin (1 µM) and metformin (50 mM) for 1 hour as comparative groups.
To perform a cell lysis assay, a sample-treated petri dish was placed on ice, a supernatant
was suctioned, and 5 mL each of a cold PBS buffer was added for washing twice. 10
µL of a protease/phosphatase inhibitor was added to 1 mL of a lysis buffer and well
mixed, and 100 µL of a cell lysis buffer was dropped on each dish, and incubated on
ice for 5 minutes. After being detached with a scraper, the cells were transferred
to a 1.5 mL microtube, and then vortexing and incubation on ice were repeated for
1 minute each for 20 minutes. Afterward, centrifugation was performed at 14,000 rpm
for 10 minutes at 4 °C, 5 µL and 70 µL of the lysed sample supernatant was transferred
to two 1.5-mL microtubes , respectively. The microtube to which 5 µL of the supernatant
was transferred was stored at -20 °C, and 16.5 µL of 5X sample buffer was put into
the microtube to which 70 µL of the supernatant was transferred and boiled at 100
°C for 5 minutes. The boiled sample was stored at -20 °C. To perform a BCA quantification
analysis, the 1.5-mL microtube to which 5 µL of the lysed sample supernatant was added
was taken out at room temperature, vortexed with 20 µL of sterile distilled water,
and then spun down. 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0 mg/mL of stocks
were prepared by dissolving 2 mg/mL bovine serum albumin (BSA) in sterile distilled
water and then diluting by 1/2. Each concentration of BSA was added at 25 µL into
3 wells in a 96 well polystyrene plate, and 25 µL of the lysed sample was also added
to one well. 8 mL of BCA Protein Assay Reagent A and 160 µL of BCA Protein Assay Reagent
B were mixed, and then 200 µL thereof was put into the wells. After a reaction in
a 37 °C incubator for 30 minutes, absorbance was measured at 562 nm using a SpectraMax
M3 microplate reader (Molecular Devices, USA). For western blotting, a 10% gel was
prepared during Tris-glycine SDS-polyacrylamide gel electrophoresis, 50 µg of a protein
per sample was measured and loaded. After transfer to a nitrocellulose membrane, the
reaction was blocked with 5% skim milk, and AMPKα antibodies were diluted 1:400, phospho-AMPKa
antibodies were diluted 1:400, and β-actin antibodies were diluted 1:1,000 with 5%
skim milk, and mixed overnight with the membrane. After the resulting membrane was
washed with a 1X PBST (0.05% Tween-20-containing PBS) solution three times for 5 minutes,
anti-rabbit IgG and HRP-linked antibodies were diluted 1:1,000, and mixed with the
membrane for 1 hour. The resulting membrane was washed with a 1X PBST solution three
times for 5 minutes, and then a solution in which solution A and solution B of the
West-Q Chemiluminescent Substrate Kit were mixed at 1:1 was sufficiently sprayed on
the membrane, followed by detecting bands using a Chemidoc system.
[0108] As a result, as shown in FIG. 4, it was confirmed that the expression of phosphorylated
AMPK increases depending on a treatment concentration of the
Lactobacillus paracasei-derived extracellular vesicles (MDH-001-CM). It can be seen that the
Lactobacillus paracasei-derived extracellular vesicles activate AMPK signaling in a dose-dependent manner, induce
autophagy and inhibit a metabolic function disorder, thereby increasing cell homeostasis.
Example 5: Evaluation of therapeutic effect of Lactobacillus paracasei-derived extracellular vesicles in rabbit model with optical disease caused by oxidative stress
[0109] It is known that oxidative stress plays an important role in various ocular diseases
such as age-related degeneration (AMD), retinopathy of prematurity, retinal light
damage, glaucoma and cataracts in pathological aspects (
Beatty S., Koh H., Phil M., Henson D., and Boulton M., "The role of oxidative stress
in the pathogenesis of age-related macular degeneration", Surv. Ophthalmol., 45(2):
115-134(2000)).
[0110] Therefore, to induce an ocular disease by oxidative stress, sodium iodate (SI), which
is a material for inducing macular degeneration by oxidative stress, was intravenously
administered into a rabbit once. To evaluate the therapeutic effect of the
Lactobacillus paracasei-derived extracellular vesicles, 0.25 mg/kg and 2.5 mg/kg of the
Lactobacillus paracasei-derived extracellular vesicles (MDH-001) were orally administered once daily 3 days before
induction to 7 days after induction. For evaluation, a retinal degenerated area was
taken with a fundus camera (TRC-50IX, TOPCON, Japan) on the final administration day,
i.e., on day 7 after induction of the disease, and analyzed (FIG. 5).
[0111] As a result, as shown in FIG. 6, in a low-dose vesicle (MDH-001)-treated group (G3)
and a high-dose vesicle (MDH-001)-treated group (G4), compared to a positive control
(G2), the degenerated area of the retinal pigment epithelium (RPE) was statistically
significantly reduced, confirming dose-dependence.
[0112] Meanwhile, to evaluate the effect of the
Lactobacillus paracasei-derived extracellular vesicles in treatment of macular degeneration, after instilling a mydriatic
agent (Midriacil 1% eye drop) into the right eye of the rabbit, the animal was anesthetized,
and then its fundus was photographed with a fundus camera. The result is shown in
FIG. 7.
[0113] As shown in FIG. 7, it was seen that almost no macular degeneration was shown in
a negative control (G1), and macular degeneration was clearly shown in a positive
control (G2) in which the disease was induced. In addition, it can be seen that, in
the low-dose vesicle-treated group (G3) and the high-dose vesicle-treated group (G4),
compared to the positive control, macular degeneration was significantly reduced,
and in the high-dose treated group, a reduction effect was more clearly shown.
[0114] From the above results, it was seen that the
Lactobacillus paracasei-derived extracellular vesicles of the present invention can effectively inhibit the occurrence
of an ocular disease by oxidative stress. Particularly, it was confirmed that causes
of cell age-related ocular diseases can be efficiently regulated by not only inhibiting
the secretion of an inflammatory mediator by a pathogenic factor but also increasing
the resistance to metabolic stress through AMPK signaling (FIG. 8).
[0115] Therefore, it is expected that the
Lactobacillus paracasei-derived extracellular vesicles of the present invention can be used for alleviating, preventing
or treating age-related and inflammatory ocular diseases.
[0116] The above-described description of the present invention is provided for illustrative
purposes, and those of ordinary skill in the art to which the present invention pertains
will understand that the present invention can be easily modified into other specific
forms without changing the technical spirit or essential features of the present invention.
Therefore, it should be understood that the above-described Examples are illustrative
only in all aspects and are not restrictive.
[industrial Applicability]
[0117] It was confirmed that, after being absorbed into the body,
Lactobacillus paracasei-derived extracellular vesicles according to the present invention are distributed in the
central nervous system through the blood brain barrier (BBB), after being absorbed
into cells, effectively inhibit the secretion of IL-6, which is the main mediator
of intractable inflammatory diseases, activate AMPK signaling to increase metabolic
function, and efficiently inhibit the occurrence of an ocular disease caused by cell
senescence and inflammation. Further, as it was confirmed that, when the vesicles
were administered into a rabbit model with an ocular disease caused by oxidative stress,
the occurrence of retinal degeneration caused by oxidative stress was significantly
inhibited, the
Lactobacillus paracasei-derived extracellular vesicles according to the present invention are able to be widely used
as a drug for alleviating, preventing or treating an ocular disease, and thus has
industrial applicability.